PROJECT SUMMARY Most quiescent and differentiated mammalian cells assemble primary cilia, antenna-like organelles that emanate from the cell surface and sense extracellular cues and growth factors. Failure to assemble and maintain normal primary cilia and centrosomes leads to developmental defects and ciliopathies. Ciliogenesis begins when the maternal centriole matures and acquires a new function as basal body, recruiting vesicles that build the ciliary membrane and allow fusion with the plasma membrane. Mechanistically, the details of this remarkable transformation remain a mystery. For example, it is not known how centrioles mature to basal bodies and how the basal body recruits vesicles needed to build the primary cilium in a manner that is coordinated with microtubule growth and axoneme elongation. Indeed, the key steps that control the early stages of cilium assembly are not well understood. Our central hypothesis states that key controls govern initiation of basal body formation and recruitment of vesicles required for cilium assembly and that these transitions critically depend on the function of a network of proteins at distal ends of basal bodies. In this proposal, we will investigate the function of early ciliogenesis regulators, and in particular, Talpid3, a protein required for ciliogenesis. We propose that Talpid3 is required to promote cilium assembly through basal body maturation and recruitment of proteins required for assembly of ciliary vesicles and will test this hypothesis in three Aims. In the first Aim, we will test a role for Talpid3 in the specification of mother centriole identity and basal body assembly by combining structure- function analyses with use of knock-out cells and loss-of-function mutants. The function of Talpid3 remains unknown, and in Aim 2, we will examine the function of Talpid3 in cilium assembly and the impact of mutations in the human gene, which result in a ciliopathy, Joubert Syndrome, using human cells and zebrafish. We have shown that loss of Talpid3 leads to defects in centriole length and assembly of distal appendages required to recruit ciliary vesicles. Using cells carrying loss-of-function and Joubert Syndrome mutations, we will explore interactions with other distal centriolar proteins and link their function to the mechanistic roles of Talpid3 in distal appendage assembly, centriole length control, ciliary vesicle recruitment, and ciliogenesis. In the final Aim, we will perform proteomic and functional siRNA screens to identify additional regulators of early ciliogenesis, allowing us to uncover the network of proteins that interact with Talpid3 to build distal appendages and anchor vesicles needed to build the cilium. Using a combination of state-of-the art imaging and cell biology, directed siRNA screens, biochemistry, and human mutation data, we will begin to link genes and mechanisms that underlie early ciliogenesis with processes that are deficient in human disease. Our studies will also unravel fundamental mechanisms that specify basal body identity during the early stages of ciliogenesis.